The overall goal of this SBIR application is to develop and commercialize a next generation allergy biologic therapy. This novel therapeutic, an anti-IgE monoclonal half antibody, targets the surface-bound IgE on allergic effector cells and functionally blocks allergic reactivity. Allergic disorders have increased to the point where they have now become a major worldwide public health issue and particularly so in developed countries such as the US. Novel effective treatment options for allergies remain a major unmet need; no effective therapy is available for severe food allergies such as peanut allergy and no new platforms for inhalant allergy have been licensed since Xolair(r)-based anti-IgE therapy in 2003. In this application we propose a new approach where a genetically engineered anti-? mAb half molecule [AEHM] E-3.56 is used to block allergic reactivity. The rationale of this therapeutic approach is based on our preliminary finding that binding of this AEHM to the Fc?RI bound IgE on allergic effector cells is incapable of mediating the cross-linking and triggering the cells due to its monovalent nature while at the same time, its binding inhibits subsequent allergen-induced reactivity. Thus the primary objective of this application is to produce a genetically engineered prototype AEHM E-3.56 and experimentally show that E-3.56 binding to surface-bound IgE on the allergic effector cells functions as a novel therapeutic against allergic reactivity. In Aim 1,we will engineer and express a prototypic IgG1 anti-epsilon (IgE) half antibody molecule E-3.56 composed of the mouse VH and VL regions linked to the human ?1 heavy chain and ? light chain constant regions respectively, with the two key cysteines in the C?1 hinge region mutated to preventing dimerization of the two CH chains. Co-expression of the chimeric H and L chains either by transient transfection or via established stable cell lines will result in the productionof E-3.56 that is monovalent for human IgE binding. In Aim 2, we will test the safety profile and therapeutic efficacy of E-3.56 for its ability to inhibit allergic responses in in vitro and in viv established allergy models. We will use a systemic allergic anaphylaxis model, passive cutaneous anaphylaxis model and human basophil stimulation to test an E-3.56's safety and therapeutic efficacy. In Aim 3 we will define by which of the following mechanisms E-3.56 suppresses allergic reactivity: 1) Steric hindrance preventing access to the allergen binding sites; 2) Triggering negative signaling in effector mast cells and basophils; 3) Promoting clearance of cell surface IgE- Fc?RI complexes; 4) Triggering conformational change of IgE's allergen-binding sites leading to loss of allergen binding; and/or 5) Interfering with the IgE cros-linking necessary for allergic signaling. This application is directed at the development of a nove allergy therapeutic that is relevant to the public health.